A tumor therapeutic vaccine designed by using nanoscale materials as the carrier is a new way to treat the tumor. Existing researches show that using the nanoscale material to carry protein antigen can increase the imnmnogenicity of the antigen. This phenomenon may relate to the relatively huge surface area and the complex structure of the nanoscale material. Meanwhile, the nanoscale material carrier can protect the protein antigen to a certain extent to prevent the protein antigen from degradation by the protease and the inner-system quickly, helping the protein antigen play its role effectively. Additionally, the nanoscale material carrier carrying the protein antigen is easy for intake and process by antigen present cells (APCs), especially dendritic cells (DCs) to make the DCs process and present a large amount of protein antigen to induce a more effective immune response. Using specific nanoscale material to entrap tumor-specific antigen and immunoadjuvant to form tumor therapeutic vaccine and inoculating the vaccine to a patient with tumor can stimulate the immune system of the patient, trigger an immune response to kill tumor cells, and reduce or eliminate the remains of tumor focuses in the body after operation or radiotherapy and chemotherapy.
Currently, although many kinds of tumor vaccine design schemes, using nanoscale materials as a carrier, have appeared, but these all face critical technical obstacles. Although the tumor vaccine using a virus, virus-like particle or virosome as a carrier (1. Arlen P M, et al. A randomized phase II study of docetaxel alone or in combination with PANVAC-V (vaccinia) and PANVAC-F (fowlpox) in patients with metastatic breast cancer (NCI 05-C-0229). Clin Breast Cancer. 2006; 7:176-179; 2. Gulley J L, et al. Immunologic and prognostic factors associated with overall survival employing poxviral-based PSA vaccine in metastatic castrate-resistant prostate cancer. Cancer Immunol Immunother. 2009) can stimulate the organism to induce an immune response at the beginning of use, the protein component of the virus itself is an effective antigen and dominates. As a result, the induced immune response is mainly aiming at the antigen of the virus carrier itself rather than the tumor specific antigen carried by the virus carrier. This effect is more obvious, especially in the situation where the antigenicity of the tumor specific antigen is relatively weak. In order to improve the immunotherapy effect of the therapeutic vaccine, normally multiple immunizations will be conducted. In this situation, the immune response (mainly the antibody response) to the virus carrier itself, induced by the previous immunization, will significantly reduce the effect of the subsequent immunotherapy. Moreover, the vaccine designed by using common virus (for example, adenovirus, poxvirus, etc.) as carrier normally has a significantly reduced immunotherapy effect due to the possible immune protection generated by the patient's early virus infection (Huang X, Yang Y. Innate immune recognition of viruses and viral vectors. Hum Gene Ther. 2009; 20:293-301.).
For nanovaccine using lipidosome as a carrier, since the carrier itself does not have immunogenicity, the immune effect of the tumor specific antigen can be improved. However, due to the size and component of the lipidosome carrier, its particle cannot effectively diffuse into the draining lymph node after the hypodermic vaccination. Some of the vaccine components remain in the vaccinating area for a long time. As a result, a large amount of tumor antigen-specific cytotoxic lymphocytes (CTLs), induced by immunization, will immigrate to the vaccinating area rather than the tumor growing area so that the specific immune response to the tumor is significantly lowered. (Hailemichael Y, et al. Persistent antigen at vaccination sites induces tumor-specific CD8+T cell sequestration, dysfunction, and deletion. Nat Med. 2013; 19(4): 465-72.) In addition, due to the characteristics of the preparation of lipidosome, the obtained product normally has poor homogeneity, large particle size range, and normal repeatability among production batches. Therefore, it exists a certain difficulty in quality control.
Micellar carrier is a kind of nanoparticles formed of self-assembling amphiphilic polymer molecules including both hydrophilic blocks and hydrophobic blocks. The assembly of the micellar nanoparticles is that the polymer molecules spontaneously form a thermodynamically stable system in an aqueous solution. This process is caused by free energy decrease due to spontaneous accumulation and polymerization of the hydrophobic blocks withdrawn from the aqueous solution. Comparing with surfactants with low molecular-weight, the critical micelle concentration (CMC) of the amphiphilic polymer is lower such that the polymer micelles can resist the dilution of the solution. Meanwhile, the micellar core formed of hydrophobic blocks has a compact structure which makes it hard to dissociate after being diluted with a large amount of body fluid. Therefore, it has a better stability. Generally, the hydrophilic blocks of the amphiphilic polymer molecule are made of polyethylene glycol (PEG), which has a good water solhbility and a high level of hydration characteristic such that it can provide sufficient steric hindrance for the micellar particle at the shell area thereof. Additionally, it has good biocompatibility and is a widely used pharmaceutic adjuvant verified by FDA. There are lots of materials that can be used for the hydrophobic blocks of the amphiphilic polymer molecule, and the material used is a key factor for the drug loading efficiency and stability of the micellar carrier. Based on the chemical structure, the lipophilic group of the hydrophobic blocks can be divided into three types, i.e., polyester derivative, poly-amino acid derivative, and pluronics types. In core blocks of the polyester type, poly-lactic acid (PLA), poly-caprolactone (PCL), and poly-glycolic acid are all materials confirmed by FDA with good biocompatibility. In core blocks of the poly-amino acid type, materials such as poly-aspartic acid (PAsp), polyglutamic acid (PGlu), poly-L-lysine (PLys), poly-histidine (Phis), and so on have been commonly used. The pluronics is the triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, and can be written as PEOm-PPOn-PEOm.
The pegylated phospholipid is a new type of amphiphilic polymer molecule. The hydrophilic blocks of the pegylated phospholipid are polyethylene glycols, and the hydrophobic blocks of the pegylated phospholipid are lipid molecules. The polyethylene glycol molecules are covalently bonded with nitrogenous bases of the lipid molecules. Currently, the pegylated phospholipid is used as an entrapping material to mainly entrap chemotherapeutic drugs having small molecules. The advantages are that: 1) the phospholipid with hydrophobic cores can entrap a poorly soluble drug to significantly improve the solubility of the drug; 2) the polyethylene glycol with hydrophilic shells can protect the drug molecules inside the micelle from being absorbed or degraded from outside to help the drug escape from intake by the reticuloendothelial system and extend the circulation time of the drug; 3) the releasing of the drug can be controlled and the intracorporal distribution of the drug can be optimized to obtain a better therapeutic effect (without causing immune response). However, there is few research about the entrapment of pegylated phospholipid micelle to protein or peptide, and no research about tumor therapeutic vaccine developed by using micelles made of the pegylated phospholipid as carrier system is reported in this country and abroad.
Monophosphoryl Lipid A (MPLA) is a common immunoadjuvant (see FIG. 9 for structure), which is a new type of immunoadjuvant obtained by chemically modifying lipopolysaccharide (LPS) derived from salmonella R595. Comparing with LPS, MPLA substantially keeps the ability of immune stimulation while significantly lowering the endotoxin toxicity. Thus, it becomes a safer and more effective immunoadjuvant, and has been granted by FDA as immunoadjuvant entering clinical. Similar to the molecular mechanism by which the LPS functions, MPLA functions by interacting with Toll-like receptor in 4 phases, activating the signal passage downstream of the MPLA that relates to natural immunization, activating natural immunization responses, promoting the expression of interferon γ and tumor necrosis factor α, while activating dendritic cells to further activate an acquired immune response.